Toad Plasma Research Articles

Day Versus Night Melatonin and Corticosterone Modulation by LPS in Distinct Tissues of Toads (Rhinella Icterica).

Pathogen-associated molecular patterns modulate melatonin (MEL) production in the pineal and extra-pineal sites and corticosterone (CORT) synthesis in the adrenal/interrenal and other tissues. Both MEL and CORT play essential and complex immunomodulatory roles, controlling the inflammatory response. Given that most of what we know about these interactions is derived from mammalian studies, discovering how MEL and CORT are modulated following an immune challenge in anurans would increase understanding of how conserved these immune-endocrine interactions are in vertebrates. Herein, we investigated the modulation of MEL and CORT in plasma vs. local tissues of toads (Rhinella icterica) in response to an immune challenge with lipopolysaccharide (LPS; 2 mg/kg) at day and night. Blood samples were taken 2 hours after injection (noon and midnight), and individuals were killed for tissue collection (bone marrow, lungs, liver, and intestine). MEL and CORT were determined in plasma and tissue homogenates. LPS treatment increased MEL concentration in bone marrow during the day. Intestine MEL levels were higher at night than during the day, particularly in LPS-injected toads. Bone marrow and lungs showed the highest MEL levels among tissues. Plasma MEL levels were not affected by either the treatment or the phase. Plasma CORT levels increased in LPS-treated individuals, with an accentuated increase at night. Otherwise, CORT concentration in the tissues was not affected by LPS exposure. Modulation of MEL levels in bone marrow suggests this tissue may participate in the toad's inflammatory response assembly. Moreover, MEL and CORT levels were different in tissues, pointing to an independent modulation of hormonal concentration. Our results suggest an important role of immune challenge in modulating MEL and CORT, bringing essential insights into the hormone-immune interactions during anuran's inflammatory response.

Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms

Is snake venom activity influenced by size? This is a long-standing question that can have important consequences for the treatment of snake envenomation. Ontogenetic shifts in venom composition are a well-documented characteristic of numerous snake species. Although snake venoms can cause a range of pathophysiological disturbances, establishing the coagulotoxic profiles related to such shifts is a justified approach because coagulotoxicity can be deadly, and its neutralisation is a challenge for current antivenom therapy. Thus, we aimed to assess the coagulotoxicity patterns on plasma and fibrinogen produced by B othrops jararacussu venoms from individuals of different sizes and sex, and the neutralisation potential of SAB (anti bothropic serum produced by Butantan Institute). The use of a metalloproteinase inhibitor (Prinomastat) and a serine proteinase inhibitor (AEBSF) enabled us to determine the toxin class responsible for the observed coagulopathy: activity on plasma was found to be metalloprotease driven, while the activity on fibrinogen is serine protease driven. To further explore differences in venom activity, the activation of Factor X and prothrombin as a function of snake size was also evaluated. All the venoms exhibited a potent procoagulant effect upon plasma and were less potent in their pseudo-procoagulant clotting effect upon fibrinogen. On human plasma, the venoms from smaller snakes produced more rapid clotting than the larger ones. In contrast, the venom activity on fibrinogen had no relation with size or sex. The difference in procoagulant potency was correlated with the bigger snakes being proportionally better neutralized by antivenom due to the lower levels of procoagulant toxins, than the smaller. Thus, while the antivenom ultimately neutralized the venoms, proportionally more would be needed for an equal mass of venom from a small snake than a large one. Similarly, the neutralisation by SAB of the pseudo-procoagulant clotting effects was also correlated with relative potency, with the smaller and bigger snakes being neutralized proportional to potency, but with no correlation to size. Thromboelastography (TEG) tests on human and toad plasma revealed that small snakes’ venoms acted quicker than large snakes’ venom on both plasmas, with the action upon amphibian plasma consistent with smaller snakes taking a larger proportion of anuran prey than adults. Altogether, the ontogenetic differences regarding coagulotoxic potency and corresponding impact upon relative antivenom neutralisation of snakes with different sizes were shown, underscoring the medical importance of investigating ontogenetic changes in order to provide data crucial for evidence-based design of clinical management strategies.

Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus.

Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Chronic blood pressure effects of bufalin, a sodium-potassium ATPase inhibitor, in rats.

Endogenous Na+,K(+)-ATPase inhibitors may have a role in the mechanism of low-renin hypertension. Two such compounds have been characterized: ouabain from human plasma and resibufogenin from toad plasma. Previously, we examined the acute effects of ouabain and bufalin (which has the same structure as resibufogenin except for one H+) in normal rats. Bufalin raised blood pressure, but ouabain had little effect. In contrast, given chronically, ouabain substantially increased blood pressure in normal rats and 70% reduced renal mass rats on a salt-free diet. We have now examined the chronic effects of bufalin in rats. Normal rats received 14.8 micrograms/kg per day bufalin or an equimolar dose of ouabain intraperitoneally for 6 weeks; 70% reduced renal mass rats also received 14.8 micrograms/kg per day bufalin. Another group of normal rats received 29.6 micrograms/kg per day bufalin intraperitoneally for 6 weeks. Respective control animals received vehicle. In contrast to ouabain, blood pressure did not increase in normal rats receiving the 14.8 micrograms dose of bufalin. However, normal rats receiving 29.6 micrograms bufalin and 70% reduced renal mass rats receiving 14.8 micrograms bufalin developed significant increases in blood pressure. Increases in blood pressure were associated with decreases in myocardial Na+,K(+)-ATPase activity and correlated with increased plasma Na+,K(+)-ATPase inhibitory activity. Thus, although bufalin is a more potent pressor agent than ouabain when both agents are given acutely, ouabain is at least as potent a vasopressor agent as bufalin when given chronically. Thus, both are pressor agents, more so in the presence of reduced renal mass, when given chronically in the rat.

Taurine in toad brain and blood under different conditions of osmolality: An immunohistochemical study

The concentrations of taurine in blood and brain regions of the toad Bufo boreas have been measured. Most of these values are considerably lower than those found in mammals. Using an antibody prepared against conjugated taurine, the distribution of taurine in three brain regions of the toad has been visualized. The possible osmoregulatory functions of taurine have been investigated by making toads hyper- or hypo-osmotic in vivo. Induction of hypoosmolality is accompanied by a massive taurine tide in blood plasma, but has no immediate effects upon the taurine concentrations in the brain areas studied. However, histochemical visualization indicates a marked redistribution of taurine between cellular components and extracellular space of brain tissues. This may indicate that taurine has an osmoregulatory function in brain tissue under hypo-osmotic conditions. Hyperosmolality results in no elevation of the taurine concentration in blood plasma of toads, but rather in a very gradual decline of total plasma taurine content over a prolonged time period. Histochemical studies reveal little change in frontal cortex after 1 hour but deeper staining of many neurons in optic lobe accompanied by greater staining in the extracellular fluid. By 3 hours there is a depletion of taurine from all compartments of cerebral cortex tissues. No evidence of any prolonged direct osmoregulatory role for taurine is indicated under hyperosmotic conditions. A possible indirect osmoregulatory function of taurine is discussed.

Digitalis-like compounds in the toad Bufo viridis: interactions with plasma proteins.

Digitalis-like compounds (DLC), normal constituents of animal tissues, are possible regulators of the Na+,K(+)-ATPase implicated in water and salt homeostasis. DLC are present in toad (Bufo viridis) tissues. Although DLC highest levels were found in toad skin, it was also detected in plasma and many internal organs. The abundant distribution and the different levels of DLC in various tissues exclude the possibility that toxicity is the only function of these compounds in the toad. The concentration of DLC in toad plasma is 30 microM, out of which 25-30% is bound to plasma proteins. Fractionation of toad plasma proteins on a G-100 Sephadex column followed by the extraction of DLC from the plasma proteins revealed that DLC are bound primarily to proteins of 48,000-53,000 Da. These results establish the existence of bufodienolide-binding protein(s) in animal plasma.

Acid-base-electrolyte balance responses of Bufo marinus to aminoglutethimide, corticosterone, and aldosterone during hypercapnia

Experiments were conducted to test the hypothesis that one or more interrenal steroids are active in regulatory responses to respiratory acidosis in the toad, Bufo marinus. Toads were divided into four experimental groups. The first group received sham injections. The second group received 1–3 mg of aminoglutethimide (AG) every 8 hr. AG inhibits the conversion of cholesterol to pregnenolone, thus inhibiting all steroid hormone synthesis. The third group received AG + 5 μg of aldosterone on the same schedule. The fourth group received AG + 25 μg of corticosterone on the same schedule as the other groups. All four groups were subjected to hypercapnia using 5% CO 2 to induce a respiratory acidosis. The sham-operated animals displayed the normal compensatory pattern of producing a metabolic alkalosis (elevated plasma HCO 3 − ) after 24 hr. AG-treated toads failed to elevate plasma HCO 3 − . Administration of interrenal steroids produced compensation in varying degrees. Aldosterone produced a small compensation while corticosterone produced a compensation similar to that seen in sham-operated animals. Analysis of steroid titers in toad plasma during hypercapnia showed that Bufo marinus does not elevate aldosterone during respiratory acidosis, but that corticosterone is elevated. AG blocked the corticosterone elevation, however. AG also produced a hyponatremia that was corrected with aldosterone or corticosterone. Normocapnic controls showed that AG does not produce deleterious effects on pH or blood gases in toads in the absence of a respiratory acidosis. We conclude that corticosterone is important in acid-base regulatory responses to respiratory acidosis in this amphibian.

Effect of salt acclimation on digitalis-like compounds in the toad

Digitalis-like compounds (DLC) were shown to be a normal constituent of the skin and plasma of toads. In order to assess the possible physiological role of these compounds in the toad, their levels were determined in the brain, plasma and skin following acclimation in different NaCl solutions. We demonstrate that an increase in salt concentrations in the animal medium from 0 to 1.2% decreased the levels of DLC in the brain by 50% without altering significantly its levels in the plasma and skin. An increase in medium salt concentration to 1.5% resulted in a 50% increase of DLC levels in the skin without changing its levels in the plasma or brain. These results suggest that skin and brain DLC may participate in the long-term salt and water homeostasis in the toad, while the plasma compound either participates in the short-term regulations of salt and water homeostasis or have some other, unknown, function.

Insulin-like growth factor-I and insulin-like growth factor-binding protein in the toad, Bufo woodhousei

Molecular weight characteristics and plasma concentrations of insulin-like growth factor-I (IGF-I) and its binding protein (IGF-BP) were investigated in the toad, Bufo woodhousei. IGF-I and IGF-BP were measured by radioimmunoassay (RIA, K d = 0.37 ± 0.04 ng/ml) and charcoal-separated ligand binding assay, respectively, in male toad plasma and adult male human donor plasma using a synthetic human IGF-I standard. Prior to the IGF-I RIA, samples were acid-ethanol extracted. Molecular weight characteristics were determined using size exclusion chromatography. At neutral pH (pH = 7.4), IGF-I immunoreactivity and IGF-BP eluted at molecular weight >66 kDa in both toad and human plasma. Acid chromatography (pH ∼3) resulted in the separation of IGF-I from its binding protein and consequently a shift of IGF-I immunoreactivity to the low molecular weight fractions (∼8 kDa) for both toad and human. IGF-BP activity shifted to molecular weight ∼50 kDa. Toad plasma IGF-I and IGF-BP activity exhibited differences according to season: IGF-I levels were low in the spring (March = 0.48 ± 0.11 ng eq/ml), increased progressively to reach a peak in July (5.84 ± 2.5 ng eq/ml), and decreased to low levels again in the fall (October = 0.60 ± 0.08, November = 0.45 ± 0.09 ng eq/ml). Plasma IGF-BP activity demonstrated a similar pattern (March = 17.4 ± 2.5, July = 35.0 ± 2.4, November = 12.6 ± 3.2% specific binding). IGF-I was produced for at least 72 hr when toad liver explants were cultured in serum-free medium, indicating that the liver is a source of IGF-I in anurans.

Plasma cathepsin activity and the role of hypovolemic shock in dehydrational death in the toad, bufo marinus

1. 1. The pH- and activator-dependence of toad plasma cathepsin activity assayed by hemoglobin digestion was characteristic of cathepsins B1 and D. 2. 2. Dehydration, even to the point of death, did not produce a significant elevation of plasma cathepsin activity over controls. 3. 3. Toads were remarkably resistant to the effects of splanchnic artery ligation, which also did not produce significantly higher plasma cathepsin levels. 4. Cardiac depression via the production of a myocardial depressant factor by cathepsins does not appear to be an important factor in dehydrational death in toads.

Identification of a ouabain-like compound in toad skin and plasma as a bufodienolide derivative

An ouabain-like compound (OLC) was purified from toad skin. The purification procedure consisted of three sequential separations on HPLC using amino and reverse phase chromatography. Using UV, NMR and Mass spectroscopy the structure of the purified material is suggested to be mono-hydroxy-14, 15-epoxy-20, 22-dienolide glycoside (resibufogenin). Evidence is presented that this compound is also present in the toad plasma. It is suggested that the endogenous bufodienolide derivative participates in the physiological regulation of the Na +, K +-ATPase activity.

Further characterisation of the inotropic effect of a bufodienolide glycoside--an endogenous ouabain like compound.

A ouabain like compound obtained from toad skin and plasma and identified to be a steroidal bufodienolide glycoside was found to displace ouabain from its binding site and to inhibit Na+-K+ ATPase, and have positive inotropic effects on cardiac muscle. The ionic and rate dependence of this positive inotropy was studied in the frog atrium. The effect was dependent on extracellular potassium, sodium, and calcium concentrations and on the rate of stimulation, which is similar to the properties of cardiac glycosides. Occasionally, the compound gave transient or even negative inotropic responses, as do the glycosides. The action potential configuration was also affected by the compound in the same complex pattern as is that of cardiac glycosides. It is concluded that the endogenous bufodienolide compound has the same physiological effects as cardiac glycosides. Since the same compound is present in toad plasma it may serve as an intrinsic humoral regulator of cardiac contractility. This study is the first detailed characterisation of the cardioactive properties of this compound.

Biosynthesis of bufadienolides in toads. V. the origin of the cholesterol used by toad parotoid glands for biosynthesis of bufadienolides

Sodium (1- 14C) acetate and (5- 3H) mevalonate were incubated with Bufo vienarum toad parotoid gland and liver tissues. Both labelled compounds were incorporated into cholesterol produced by liver while the incubations with parotoid gland produced no labelled cholesterol. Low and high density lipoproteins isolated from toad plasma were iodinated and used for binding studies. Membrane preparations of paratoid gland showed high affinity binding sites for 125I-LDL and 125I-HDL. In addition, while colchicine inhibits the in vitro uptake of ( 3H) cholesteryl linoleate-LDL into parotoid gland tissue an opposite effect was seen with ( 3H) cholesteryl linoleate-HDL. The above mentioned results would support the hypothesis that the cholesterol used by the parotoid gland for the biosynthesis of bufalienolides would be produced in the liver, transported by the circulating lipoproteins and incorporated by the glands by a receptor-mediated mechanism.

Anion permeability of toad skeletal muscle incubated in plasma and ringer solution.

Isolated semitendinosus muscles of the toad were incubated in both Ringer solution and toad plasma for periods ranging from 45 to 170 minutes. The extracellular volume (ECV), as measured from the equilibrium distribution of 35SO4(2-), was found to increase (in gram extracellular (EC) water/gram wet weight) from 0.15 +/- 0.01 to 0.20 +/- 0.02 in muscles incubated in Ringer solution, whereas the ECV was found to remain constant in tissues incubated in plasma (0.16 +/- 0.01 to 0.17 +/- 0.01), for periods up to 170 minutes. Since the true ECV, as measured morphometrically, was found to remain unchanged for muscles incubated in Ringer solution for up to 3 hours, the data for these muscles were interpreted to represent a change in anion permeability of the cellular membranes in response to the absence of some plasma fraction from the Ringer solution (with an electrolyte composition and pH similar to that of plasma). When toad plasma albumin was added to the Ringer solution, in physiological concentrations, the ECV of the muscles was observed to remain relatively constant during the 3-hour incubation period. This albumin effect on anion permeability was found to be albumin concentration-dependent. Further, other sources of albumin were found to have varying degrees of effect on the anion permeability of toad muscle, with bovine albumin serving as the most acceptable substitute for toad albumin, when incubating semitendinosus muscles.

Determination of copper concentration in blood plasma and in ocular and cerebrospinal fluids using graphite furnace atomic absorption spectroscopy

Published values for the concentration of Cu in cerebrospinal and intraocular fluids cover a very wide range (0.016 to 1.0 μg/ml) and include values which are several times higher than those which would be consistent with normal physiology. An atomic absorption spectrophotometer equipped with a graphite furnace was used to measure the Cu concentration in these fluids and in blood plasma of toads, rabbits, and cats. Under standard conditions, these fluids yielded high background absorbance and only fractional recovery of added Cu. Parameters were therefore established which eliminated both the high background and the matrix interference and allowed the determination of Cu in 10-μl aliquots of diluted blood plasma and undiluted cerebrospinal and ocular fluid samples. Under these conditions the Cumeasured in the ocular (0.011 to 0.032 μg/ml) and cerebrospinal fluids (0.033 to 0.050 μg/ml) of these three species was lower than most previously reported values and only a small fraction (1–3%) of the concentration of Cu in the plasma of the same animals (0.85 to 1.22 μg/ml).

Chloride self-exchange in toad skeletal muscle in vivo and in vitro.

The exchange of cellular Cl with 36Cl has been measured in saline-perfused hindlimb muscles of the pithed toad and compared with cellular Cl exchange in isolated muscles incubated in the vitro either in toad Ringer solution or in toad plasma. In the perfused hindlimb, the rate of 36Cl efflux from muscle cells [17.0 +/- 0.9 pmol Cl.(cm2 plasma membrane.s)-1] was only 40% as fast as that of the 36Cl influx. The discrepancy between Cl influx and efflux was accompanied by a cellular accumulation of Cl against the electrochemical gradient for this anion. Concurrently, the cells took up Na in amounts at least equal to the accumulated Cl. During this accumulation of Na and Cl, the mean resting potential remained constant at a value of -89.2 +/- 1.9 (SE) mV. Na and Cl were taken up by the muscle cells of the perfused hindlimb without a concomitant decrease in cellular K content; i.e., without evidence of inhibition of the Na-K pump. The rate constant for cellular 36Cl efflux from isolated toad muscles preincubated for 3 h in vitro in toad Ringer solution was about five times faster than that of muscles in the perfused hindlimb and similar in magnitude to published values for Cl fluxes in frog muscle. Cellular Cl efflux from muscles briefly preincubated in vitro for 15 min instead of 3 h was significantly slower than after prolonged preincubation. In vitro incubation of isolated toad muscles in toad plasma slowed the cellular 36Cl efflux to values approaching those measured in the perfused hindlimb, without comparably depressing the 36Cl influx. It is suggested that the uptake of NaCl by the cells of perfused hindlimb muscle may proceed by an electroneutral inward cotransport of Na and Cl on the same carrier.

Is chloride passively distributed in skeletal muscle in vivo?

Boyle and Conway (1941) and Hodgkin and Horowicz (1959) first provided evidence that chloride, CI, is passively distributed in frog skeletal muscle in vitro. This conclusion has been supported by chemical measurements of intracellular C1 concentration, [Cl]i , (Adrian, 1961) and direct measurements of intracellular C1 i (Bolton and Vaughan-Jones, 1977; activity, aCl Baumgarten and Fozzard, 1978). On the other hand some chemical measurements and electron microprobe analysis of [Cl]i (Harris, 1965; Somlyo et i al., 1977), as well as some measurements of aCl (Kernan et al., 1974; Armstrong et al., 1977) suggest that muscle cells contain more C1 than expected for a passive distribution, and i~ly that C1 is actively accunulated by the cells. To date the issue remains unresolved for muscles studied in vitro. Recently, using improved methods for determining C1 content, ~lacchia et al. (1978) have shown that [Cl]i of both frog and toad semitendinosus muscles studied in vivo is much lower than reported for these preparations in vitro. [Cl]i was as expected from a passive distribution assuming that in vivo membrane potential (F m) is similar to that found in vitro and tha t intraand extracellular activity coefficients are equal. Here we report the in vivo determination of Em' ac li , and [Cl]i in toad semitendinosus muscle. These measurements provide further evidence for the passive distribution of Cl. Toads, Bufo marinus, were pithed, the semitendinosus muscle partially exposed by blunt dissection, and a small 'Nell" created around the exposed portion. The exposed muscles were superfused at 10 ml/min with Ringer ' s so lu t ion (temperature 21-23 ~ which was cont inuously bubbled with 100% 02 . The composition of the Ringer's solution was (in n~4): 70 NaCI; 3.I KCI; 2.0 CaCI 2 ; 0.5 MgCI 2 ; 45 Na methane sulfonate; 5.0 HEPES (N-2-hydroxyethylpiperazine-N' -2ethanesulfonic acid); and 5.0 glucose. The pH was adjusted to 7.5 with approximately 2 r~l NaOH. The muscle preparation was thus superfused by an isotonic solution whose Na, K, and C1 concentrations and pH were matched to the average composition of toad plasma (Danielson, 1964; Macchia et 8/., 1978; Macchia and Page, unpublished observations) and at the same time was perfused through the vascular system of the toad. 1 determined with C1 ion-selective was microelectrodes (Walker, 1971) made using Coming 477515 liquid ion-exchange resin (Coming Glass, Medfield, MA) and dimethyldichlorosilane to render the glass hydrophobic. The ion-selective microelectrodes were calibrated both before and after the experiment in pure solutions of KCI (i-300 m~0 and Na methanesulfonate (5-300 raM). each preparation, _ @I was calculated accordFor ing to the equations of Walker (1971) from the average potentials of the ion-selective and conventional electrodes. After the electrophysiological measurements, both semitendinosus muscles were excised. The [Cl]i of each muscle was calculated from the wet and dry weights of the muscle, water and C1 content of the plasma, the muscle C1 content, and the extracellular volume, ECV ~4acchia, et al. , 1978). The ECV of each specimen was estimated from

Evidence that a plasma factor(s) stimulates H+ and NH4+ excretion in the toad urinary bladder.

SummaryH+ excretion in the toad urinary bladder is increased during metabolic acidosis. A factor can be extracted from plasma of acidotic toads which stimulates H+ and NH4+ excretion by the toad urinary bladder. A factor has also been obtained from plasma of an acidotic dog which stimulates H+ and NH4+ excretion by the toad bladder. The solubility characteristics along with the concentration of the lipid fraction in the media suggest that this factor is hormonal in nature.

The renin-angiotensin system in Bufo arenarum and Bufo paracnemis

1. 1. Toads have in their kidneys a renin-like enzyme and in their plasma an angiotensinogen-like substance. The incubation of toad plasma with kidney extracts gives rise to a pressor substance having properties similar to angiotensin. 2. 2. The renin content of the toad kidney expressed/g of kidney is higher in smaller animals. 3. 3. Male toads have almost twice the amount of renin found in the kidney of female toads of a similar weight. 4. 4. No seasonal variation in kidney renin was observed in male toads of the same weight studied through the year. 5. 5. Toad angiotensinogen releases angiotensin with fish renin, but not with mammalian renin. Toad renin releases angiotensin with chicken plasma but not with mammalian plasma.

Seasonal variations of plasma adrenaline and noradrenaline in toads

Adrenaline and noradrenaline concentrations in plasma of toads were measured by use of a fluorimetric method. Seasonal variations in the plasma concentration of these catecholamines were verified. Adrenaline decreased markedly during the mating season. Towards the end of summer a progressive increase took place, reaching high values during autumn and winter. Maximal concentrations were found to coincide with the premating period. Noradrenaline decreased during autumn. No changes were observed in the other seasons. The possible correlation of these results with the seasonal variation of adrenal catecholamines in the toad is discussed. The mean annual concentration of adrenaline in plasma was 0.59 ≠ 0.04 μg per 100 ml, and that of noradrenaline was 0.67 ≠ 0.05 μg per 100 ml.